A gain-of-function TBX20 mutation causes congenital atrial septal defects, patent foramen ovale and cardiac valve defects

Abstract

Background: Ostium secundum atrial septal defects (ASDII) account for approximately 10% of all congenital heart defects (CHD), and mutations in cardiac transcription factors, including TBX20, were identified as an underlying cause for ASDII. However, very little is known about disease penetrance in families and functional consequences of inherited TBX20 mutations.

Methods: The coding region of TBX20 was directly sequenced in 170 ASDII patients. Functional consequences of one novel mutation were investigated by surface plasmon resonance, CD spectropolarymetry, fluorescence spectrophotometry, luciferase assay and chromatin immunoprecipitation.

Results: We found a novel mutation in a highly conserved residue in the T-box DNA binding domain (I121M) segregating with CHD in a three generation kindred. Four mutation carriers revealed cardiac phenotypes in terms of cribriform ASDII, large patent foramen ovale or cardiac valve defects. Interestingly, tertiary hydrophobic interactions within the mutant TBX20 T-box were significantly altered leading to a more dynamic structure of the protein. Moreover, Tbx20-I121M resulted in a significantly enhanced transcriptional activity, which was further increased in the presence of co-transcription factors GATA4/5 and NKX2-5. Occupancy of DNA binding sites on target genes was also increased.

Conclusions: We suggest that TBX20-I121M adopts a more fluid tertiary structure leading to enhanced interactions with cofactors and more stable transcriptional complexes on target DNA sequences. Our data, combined with that of others, suggest that human ASDII may be related to loss-of-function as well as gain-of-function TBX20 mutations.

Figure 1

(A) The relevant sequence electropherogram of TBX20 (NM_020417) exon 2 in the proband. (B) The affected amino acid (Ile121) lies in a highly conserved N-terminal region of the DNA binding T-box region of TBX20. Affected region of TBX20 homologues and human TBX paralogues are shown. (C) Family pedigree of mutation carriers. The proband is marked with an arrow. All subjects which were genotyped for TBX20-I121M are indicated with + (carrier) or − (non-carrier). Subject II:2 was presumed positive for TBX20-I121M (plus symbol in parentheses) as indicated by the genotypes of progeny (III:1 and III:2).

Figure 2

(A) The homology model of TBX20, based on the structure of human TBX3, suggests that the side chain of Ile121 packs between the side chains of Arg127 and Tyr267 and adjacent to Thr269. (B) In TBX3, the residues equivalent to Arg127 and Tyr267 (Arg130 and Tyr264) make important interactions with the DNA, making it possible that changes at residue 121 might affect the binding of the T-box to DNA.

Figure 3

Biophysical characterisation of the I121M variant T-box. (A) Far UV CD spectra of WT (solid line) and I121M variant (dashed line) TBX20 T-box domains indicate similar folded, β-sheet rich secondary structures. (B) Proportion of secondary structure folded as protein is heated. The I121M variant (dashed line) domain displays a ∼2°C reduction in T_m compared to WT (solid line), as measured by loss of CD signal at 215 nm. (C) Binding curves for I121M (dashed line) and WT (solid line) from representative surface plasmon resonance kinetic experiment. The mutation does not significantly affect the affinity of the domain for the T-site, as evidenced by very similar rates for binding and dissociation from the DNA displayed by both domains. (D) ANS fluorescence measured for I121M at 20°C and 37°C (solid and open triangles, respectively) and WT TBX20 T-box at 20 and 37°C (solid and open squares, respectively). Both domains show increased ANS binding at 37°C, a sign that tertiary contacts are relatively weak but the variant binds more ANS than the WT domain, indicating that the mutation destabilises the hydrophobic core further.

Figure 4

Transcriptional activity of WT and mutated TBX20 (I121M) and of the previously published TBX20 mutations I152M and Q195X. (A) Fold activation of a luciferase reporter construct carrying the proximal promoter elements of the Nppa gene after cotransfection of COS7 cells with expression constructs for WT and mutant TBX20 (short isoform). (B) Activation of the Nppa promoter in the presence of mutant and WT TBX20a (full length isoform), NKX2-5 and GATA4. (C, D) Fold activation of a luciferase reporter carrying proximal promoter elements of the Gja5 gene.

Figure 5

Immunoprecipitation assay with transiently transfected COS7 cells using an antibody against TBX20 and NKX2-5, followed by semiquantitative and real-time PCR reveals an enhanced occupancy of the Nppa promoter. Fold enrichment relative to WT Tbx20 was calculated using the comparative CT method. HPRT unrelated negative control; Rabbit IgG: negative control antibody. Lower panel: schematic diagram of the proximal Nppa promoter indicating the positions of T-box, GATA, and Nkx2 factor binding sites. The positions of the primers used for the ChIP analysis are indicated as arrowheads.